Method for improved protein production in filamentous fungi

ABSTRACT

The present invention relates to a method for genetically modifying a filamentous fungus host for improved protein production. The method comprises that a filamentous fungus host is genetically modified to overexpress or to be deficient of specific genes. The invention relates also to the modified hosts. Furthermore, the invention relates to a method for improved production or for producing an improved composition of proteins, such as cellulases, hemicellulases, other proteins involved in the degradation of lignocellulosic material, or other proteins, in a filamentous fungus host.

PRIORITY

This application is a divisional application of U.S. application Ser.No. 13/701,919 filed on Dec. 14, 2012 and claiming priority ofinternational application PCT/FI2011/050495 filed on May 30, 2011, andof Finnish national application number FI20105633 filed on Jun. 4, 2010.The content of all the priority applications is incorporated herein byreference.

SEQUENCE LISTING

This application contains sequence data provided on a computer readablediskette and as a paper version. The paper version of the sequence datais identical to the data provided on the diskette.

FIELD OF THE INVENTION

The present invention relates to a method for genetically modifying afilamentous fungus host for improved protein production. The inventionrelates also to providing a genetically modified filamentous fungus hostfor improved protein production, in particular a Trichoderma host.Furthermore, the present invention relates to a method for improvedprotein production or for producing an improved composition of proteinsin filamentous fungi. The proteins may be endogenous proteins, such ashydrolytic enzymes, or heterologous proteins.

BACKGROUND OF THE INVENTION

Many of the biopolymer degrading hydrolytic enzymes, such as cellulases,hemicellulases, ligninases and pectinases have received attentionbecause of their potential applications in food, feed, textile, and pulpand paper industries. Industrial filamentous fungi production strains,in particular Aspergillus and Trichoderma strains, can produce highamounts of extracellular enzymes. These fungi are easy and inexpensiveto grow in large bioreactors and they possess good secretion capacitycapable of carrying out similar type of protein modifications as occursin many higher eukaryotes. The existence of hypersecreting strains andstrong promoters, such as cellulase promoters, make filamentous fungihosts also potential for heterologous protein production.

It is known that the production of cellulases, hemicellulases,ligninases and pectinases are mainly regulated at the transcriptionallevel in filamentous fungi (Aro et al. FEMS Microbiology Reviews 29(2005) 719-739). Stricker et al. Appl. Microbiol. Biotechnol. (2208)78:211-220 have described the similarities and differences in thetranscriptional regulation of expression of hemicellulases andcellulases in Aspergillus niger and Hypocrea jecorina (T. reesei),including the action of XlnR and Xyr1. In Hypocrea jecorina someregulatory components function in cellulase regulation positively (XYRI,ACE2, HAP2/3/5) and some negatively (ACEI, CREI) (Kubicek et al.Biotechnology and Biofuels 2009, 2:19; Nakari-Setälä et al. Appl andEnvironmental Microbiology, July 2009, p. 4853-4860).

Although the action of some regulatory genes on the production ofcellulases and hemicellulases has been disclosed in the prior art, thereis still a need for improved strains capable of enhanced or alteredproduction of cellulases or hemicellulases or other hydrolytic enzymesin filamentous fungi.

SUMMARY OF THE INVENTION

One object of the invention is to provide a method for geneticallymodifying a filamentous fungus host for improved protein production.

Another object of the present invention is to provide geneticallymodified filamentous fungus hosts for improved protein production.

One further object of the invention is to provide a method for improvedprotein production or for producing improved composition of proteins infilamentous fungi.

In one aspect the invention provides a method for genetically modifyinga filamentous fungus host for improved protein production. The methodcomprises

-   -   genetically modifying a filamentous fungus host to overexpress        (with increased amount or activity) genes causing increased        production of cellulases, hemicellulases, other proteins        involved in degradation of lignocellulosic material and/or other        proteins;        and/or    -   genetically modifying a filamentous fungus host by making        deficient (with reduced or lacking amount or with reduced or        lacking activity) genes causing increased production of        cellulases, hemicellulases, other proteins involved in        degradation of lignocellulosic material and/or other proteins.

In a filamentous fungus host one or more of the genes causing increasedproduction of cellulases, hemicellulases, other proteins involved indegradation of lignocellulosic material and/or other proteins can begenetically modified alone or in combination.

In one aspect the invention provides a method for increasing theproduction of a set of proteins, typically secreted proteins, orproteins produced under the control of promoters of genes encodingsecreted proteins.

In another aspect the invention provides a method for reducing theproduction of a set of proteins, typically secreted proteins, in orderto modify the pattern of produced proteins or reduction of theproduction of unwanted side-products when producing e.g. heterologousproteins.

One or more of the genes causing increased production of cellulases,hemicellulases, other proteins involved in degradation oflignocellulosic material and/or other proteins can be geneticallymodified alone or in combination in a filamentous fungus host.

In various embodiments of the invention the host can be selected fromthe group comprising Trichoderma, Aspergillus, Fusarium, Neurospora,Talaromyces, Phanerochaete, Chrysosporium and Penicillium. In onespecific embodiment the filamentous fungus host is a Trichoderma host.

In one aspect of the invention the overexpressed gene causes increasedproduction of cellulases, hemicellulases, other proteins involved indegradation of lignocellulosic material and/or other proteins, typicallysecreted proteins or proteins produced using the promoters of genesencoding secreted proteins as compared to the parent host. The increasedproduction by the genetically modified hosts may be detected either ashigher maximal production level during the cultivation as compared tothe production level of the parental host or by higher production levelat any of time points of the cultivation resulting in faster productionprocess as compared to the parental host.

In one embodiment of the invention the overexpressed gene may beselected from the group comprising Trichoderma genes tre66966 (SEQ IDNO:1), tre112524 SEQ ID NO:2), tre123668 (SEQ ID NO:3) and tre120120(SEQ ID NO:5); or is the closest homologue of at least one of said genesin Aspergillus, Fusarium, Neurospora, Talaromyces, Phanerochaete,Chrysosporium or Penicillium; or a fragment or derivative of any of saidgenes or other sequence hybridizing under stringent conditions to atleast one of said genes or said homologues. The overexpression of thesegenes causes increased production of proteins, typically secretedproteins and/or proteins produced under the promoters of genes encodingsecreted proteins, proteins involved in degradation of lignocellulosicmaterial, in particular cellulases and/or hemicellulases, as compared tothe parental host.

In various embodiments of the invention filamentous fungi hosts can beconstructed overexpressing a specific gene or a combination of specificgenes, or being deficient of a specific gene or a combination ofspecific genes, or modified otherwise to alter the amount or activity ofthe protein product of the gene. In further embodiments, filamentousfungus hosts may be constructed overexpressing a specific gene or acombination of specific genes, and at the same time being deficient of aspecific gene or a combination of specific genes.

In one further aspect the invention provides a method for improvedproduction or production of an improved composition of proteins in afilamentous fungus host, which comprises genetically modifying afilamentous fungus host as described above, and growing (cultivating)the modified filamentous fungus host under suitable culture conditionsfor protein production.

In one embodiment of the invention, the produced protein product may bean endogenous enzyme. Examples of suitable enzymes are hydrolyticenzymes, in particular cellulases, hemicellulases, cellulose orhemicellulose side chain cleaving enzymes, lignocellulose degradingenzymes, in particular pectinases and ligninases; amylolytic enzymes;proteases; invertases; phytases, phosphatases and hydrophobins.

In another embodiment of the invention, the protein is a heterologous orrecombinant protein produced under the regulation of the promoter of agene that is affected by the genetical modification of the host, such ascellulase or hemicellulase promoter.

In one still further aspect the invention provides a geneticallymodified filamentous fungus host.

The host may be selected from the group comprising Trichoderma,Aspergillus, Fusarium, Neurospora, Talaromyces, Phanerochaete,Chrysosporium and Penicillium. More specifically the host may be aTrichoderma host.

By genetically modifying the regulatory genes or their regulatorymechanisms it is possible to improve the production of extracellularproteins in general, or the production of different sets of proteins andenzymes produced by filamentous fungi, in particular Trichoderma. Thesegenetic modifications can be applied also to improve production ofheterologous proteins when promoters and/or regulatory elements of genesencoding secreted proteins are used for the heterologous or recombinantexpression. The fungus host can be genetically modified to express theregulatory gene more or less abundantly or to produce more or lessactive regulatory protein from the gene. The genetic modification caninclude overexpression, deletion or any other genetic modification toalter expression strength of the gene or the activity of the product ofthe gene. The genetical modifications result in a desired effect on theproduced protein pattern by the fungal production host. It may bebeneficial to genetically modify the production hosts in such a way thatproduction of unwanted side products is reduced, or in such a way that aselected protein or a set of proteins are more abundantly expressed andother proteins produced less abundantly. Corresponding geneticmodifications can be done also in other filamentous fungi, in order tomodify protein production properties of the host, by modifying thecorresponding homologous genes. In addition the corresponding genes fromother fungal species can be introduced, as such or in a modified form,to other fungal species to get the desired effect on protein productionin the other organism.

In the following text, the invention will be further described with theaid of a detailed description and with reference to some workingexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-5 present the results of protein production by strainsgenetically modified to overexpress a specific gene.

FIG. 6A Biomass (g/l) in T. reesei precultures before induction ofhydrolytic enzyme production and in the uninduced control cultures atthe sampling time points of the induction experiment.

FIG. 6B pH of the T. reesei cultures induced either with Avicel, spruce,wheat straw or sophorose, as well as the precultures before inductionand the uninduced control cultures.

FIG. 7A. Transcript levels of a set of known genes encoding hydrolyticenzymes during an induction experiment: abf1 (arabinofuranosidase 1),bga1 (beta-galactosidase 1), bgl1 (beta-glucosidae 1), bxl1(beta-xylosidase 1), cip1 (cellulose-binding), cip2 ( ) egl1(endoglucanase 1), girl (glucuronidase 1), man1, xyn2 and xyn4.

FIG. 7B Transcript levels of a set of known genes encoding hydrolyticenzymes during an induction experiment: abf1 (arabinofuranosidase 1),bga1 (beta-galactosidase 1), bxl1 (beta-xylosidase 1), cip2 ( ), glr1(glucuronidase 1), and xyn2.

FIG. 8 Schematic view of the plasmid constructs made for transformingthe strain T. reesei QM9414 and generating the strains overexpressingthe genes encoding putative regulatory factors. The gene is inserted inthe plasmid vector by replacing the region between attR1 attR2 sitescontaining the genes ccdB and CmR with the gene specific sequences. Thenames of the plasmids and the corresponding T. reesei strains obtainedby transformation and the corresponding gene inserted in the plasmid arelisted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to improved production of various proteinsin filamentous fungi. The invention is based on the finding ofregulatory factors which affect the production of various proteins, inparticular hydrolytic enzymes. Within the scope of the present inventionare cellulases, such as cellobiohydrolases, endoglucanases andβ-glucosidases; and hemicellulases, such as xylanases, mannases,β-xylosidases; and side chain cleaving enzymes, such as arabinosidases,glucuronidases, acetyl xylan esterases; and other lignocellulosedegrading enzymes, in particular pectinases, such as endo- andexopolygalacturonases, pectin esterases, pectin and pectin acid lyase;and ligninases, such as lignin peroxidases, Mn peroxidases, laccases;amylolytic enzymes, such as α-amylases, glucoamylases, pullulanases,cyclodextrinases; hydrophobins; proteases (serine, aspartic, glutamic,metallo proteases, acidic, alkaline); invertases; phytases;phosphatases, and hydrophobins.

By overexpressing specific genes and/or by making deficient specificgenes which encode the regulatory factors in the fungal host, it ispossible to increase or alter the production of endogenous proteins, inparticular hydrolytic enzymes, such as cellulases, hemicellulases,lignocellulose degrading enzymes, other proteins involved in thedegradation of lignocellulosic material or other proteins, typicallysecreted proteins. It is also possible to produce heterologous orrecombinant proteins under the regulation of the promoters of theaffected genes, such as cellulase or hemicellulase gene promoters, orpromoters of other genes encoding proteins involved in the degradationof lignocellulosic material or other secreted protein promoters, by goodyield in the modified host.

Described herein are methods used in modifying filamentous fungi hosts,such as Trichoderma, Aspergillus, Fusarium, Neurospora, Talaromyces,Phanerochaete, Chrysosporium and Penicillium. In these methods are usedgenes selected based on expression of the genes in cultures grown ondifferent substrates and by sequence data of the genes. The sequencepreferably comprises a sequence domain characteristic for genes encodingregulatory proteins, such as transcription factors, othertranscriptional regulators, protein kinases, proteins involved inhistone modification or chromatin remodelling, or the genes arepreferably co-regulated with cellulase or hemicellulase genes in thegenome of a filamentous fungus host.

“Overexpression of a gene” (here in particular a regulating gene) can becarried out for example by introducing into a fungus host an additionalcopy or copies of a specific gene, or expressing the gene under anotherpromoter resulting in increased expression of the gene, or otherwisegenetically modifying the fungus host so that either the gene is moreabundantly expressed or the activity of the gene product is increased.The effect of overexpression of a gene on protein production can bestudied by culturing the modified host under conditions suitable forprotein production. The effect on the production of an endogenousprotein or proteins can be studied by determining for example a specificenzyme activity, determining the amount of total protein, or determiningthe amount of specific endogenous or heterologous protein produced.

“Making deficient of a gene” means either a genetic modification of thefungus host to delete or truncate a specific gene (here in particular aregulating gene) or a genetic modification of the fungus host resultingin reduced or lacking expression of the gene or reduced or lackingactivity of the gene product by any suitable method. By “inactivation”is meant a genetic modification (usually deletion) resulting in completeloss of activity of a gene product. In this invention, the effect of thegenetic modification of a specific gene on protein production can bestudied by determining for example a specific enzyme activity,determining the amount of total protein, or determining the amount ofspecific endogenous or heterologous protein produced.

By “a regulatory gene” is meant here a gene whose function has an effecton production of proteins by the fungal host. “Overexpression of thegene” (as described above) or “making deficient of a gene” (as describedabove) has an effect on protein production by the fungus. The gene canencode for example a transcription factor, other transcriptionalregulator, a protein kinase, a protein involved in histone modificationor chromatin remodelling, or other regulatory protein.

By “inducing substrates” are meant here substrates capable of inducingthe production of hydrolytic enzymes or lignocelluloses degradingenzymes, such as cellulase or hemicellulase, other protein involved inthe degradation of lignocellulosic material, or other proteins,typically secreted proteins, or proteins produced using promoters ofgenes encoding secreted proteins. For the purpose of studying the genesencoding the mentioned enzymes, for example substrates, such as Avicel®,pretreated wheat straw, pretreated spruce, lactose, spent grain extractor sophorose, or other plant derived carbon sources, can be used.Pretreatment of spruce and wheat can be carried out by using steamexplosion and washing the treated material. The fibrous fraction of thematerial can be used for the induction.

In one aspect improved production may mean improved production of adesired enzyme or other protein. As disclosed herein a filamentousfungus host may be constructed to overexpress a specific regulatory geneor genes, or may be constructed to be deficient in a specific otherregulatory gene or genes, in order to improve the protein production.

By “suitable culture conditions for protein production” is meant hereany culture conditions suitable for producing a desired protein or acombination of desired proteins. Conditions for producing hydrolyticenzymes or lignocelluloses degrading enzymes, such as cellulase orhemicellulase, other protein involved in the degradation oflignocellulosic material or for many secreted or other proteins, arewell known for a person skilled in the art.

By “improved production” is here meant in one aspect increased amount ofprotein produced. The protein may be produced into the culture medium orinto the host cell, preferably into the culture medium. Increasedproduction may be detected for example as higher maximal level ofprotein or enzymatic activity, such as cellulase or hemicellulaseactivity, or total extracellular protein produced as compared to theparent host. In addition, or alternatively, improved protein productionmay be detected as a higher level of produced enzymatic activity orprotein produced at any time point of cultivation which results inhigher production level at earlier stages in cultivation and thus infaster production process as compared to parent host strain. Improvedproduction may mean also increased production of secreted protein orenzymatic activity per biomass amount in the culture. Protein productionby a lower amount of biomass is beneficial due to easier downstream-processing of the protein product and reduced consumption ofnutrients during the production process. Also, a desired effect of thegenetic manipulation of the production strain is lowered viscosity ofthe production culture due to e.g. lowered biomass amount in theproduction process or due to other properties of the strain.

Cellulase and hemicellulase activities can be measured using a varietyof methods using different substrates (for examples of the methods, see:Zhang Y. H., Hong J., Ye X., Cellulase assays, Methods Mol. Biol.,(2009), 581:213-231; Sharrock K. R., Cellulase assay methods: a review,J. Biochem. Biophys. Methods. (1988), 17:81-105; T. K. Ghose,Measurement of cellulase activities, (1987), Pure & Appl. Chem., 59,257-268; T. K. Ghose and V. S. Bisaria, Measurement of hemicellulaseactivities. Pure & Appl. Chem., (1987), 59, 1739-1752). Cellulaseactivity can be measured e.g. as enzymatic activity against thesubstrate, 4-methylumbelliferyl-β-D-lactoside (MULac). Methods formeasuring the combined activity of CBHI, EGI and β-glucosidase (referredhere as “total MULac” activity), as well as the separate activities ofthe enzymes, using MULac as substrate have been described (Bailey andTähtiharju, 2003; Collen et al., 2005; van Tilbeurgh et al., 1982, 1985,1988). Other substrates often used for cellulase activity measurementsinclude e.g. CMC cellulose, hydroxyethylcellulose and filter paper. Thehemicellulase activity can be measured e.g. as activity against thebirch xylan substrate (Bailey et al., 1992, Bailey M. J., Biely, P. andPoutanen, K. (1992) Interlaboratory testing of methods for assay ofxylanase activity. J. Biotechnol. 23: 257-270), and production of totalextracellular protein by using any of the methods for measurement ofprotein concentration known in the art, for example using Bio-RadProtein Assay (Bio.Rad) Growth and progress of the cultivation offilamentous fungi can be determined by measuring the production ofbiomass and by measuring the pH of the culture medium. Induction ofprotein production, and differences in gene expression level can beanalysed by isolation of RNA and subjecting the samples to micro arrayhybridisation analysis or Northern hybridisation or TRAC analysis(Rautio, J. J., Smit, B. A., Wiebe, M., Penttilä, M. & Saloheimo, M.2006. Transcriptional monitoring of steady state and effects ofanaerobic phases in chemostat cultures of the filamentous fungusTrichoderma reesei. BMC Genomics 7, article number 247. 15 p.10.1186/1471-2164-7-247).

Improved CBHI activity may be detected by higher production level ofenzyme activity against MULac substrate using a method modified foranalysis of CBHI activity.

Improved EGI activity may be detected by higher production level ofenzyme activity against MULac substrate and especially by higheractivity against MULac substrate under conditions measuring specificallythe activity of EGI.

The increased hemicellulase production may be detected by higherproduction level of enzyme activity against birch xylan substrate.

In one embodiment the protein can be an endogenous protein, inparticular a hydrolytic enzyme, such as cellulase, hemicellulase orlignocellulose degrading enzyme, or other secreted protein. Morespecifically the protein can be a cellulase or hemicellulase.

In another embodiment the protein may be any protein which is producedunder the promoter of the affected endogenous genes. The protein may beproduced for example under various cellulase or hemicellulase genepromoters, such as promoters of genes encoding CBHI, EGI or XYNI.

Improved protein production may mean altered content of the proteinsproduced by the fungus host, and production of a desired protein or aprotein mixture. As disclosed herein a filamentous fungus host may beconstructed to overexpress a specific regulatory gene or genes, or maybe constructed to be deficient in a specific other regulatory gene orgenes, in order to alter the protein production compared to the parentalhost.

By the term “endogenous proteins” are meant here proteins which arenatural products of a filamentous fungus host.

By “a heterologous protein” is meant a protein that is not a naturalproduct of the fungal species.

By “recombinant proteins” are meant here proteins that are not naturalproducts of a filamentous fungus or that are produced by a non-naturalconstruction in a filamentous fungus. DNA sequences encoding desiredhomologous or heterologous proteins may be transferred by a suitablemethod to a host.

By “secretable protein” or “secreted protein” is meant here a proteinthat is secretable or secreted outside of the host cell to the culturemedium.

By increased protein production is meant protein production which is aleast 3%, preferably at least 5%, more preferably at least 10%, stillmore preferably at least 20%, still more preferably at least 30% or mostpreferably at least 50% better than protein production by using theparent fungal host strain which has not been genetically modified.

By reduced protein production is meant protein production which is atleast 3%, preferably at least 5%, more preferably at least 10%, stillmore preferably at least 20%, still more preferably at least 30% or mostpreferably at least 50% lower than protein production by using theparent fungal host strain which has not been genetically modified.

One embodiment of the invention comprises the expression of genesequences responsible of regulating the production of hydrolyticenzymes. The genes may increase or decrease the enzyme production, orthey may increase the production of some enzyme activities and decreaseother enzyme activities.

“Genetical modification” of a filamentous fungus host means here anygenetic modification method by which a filamentous fungus host ismodified to overexpress a specific regulatory gene or to gain modifiedproperties of the gene or genes and/or to be deficient of the gene orgenes.

Genetical modification methods for the strains of Trichoderma,Aspergillus, Fusarium, Neurospora, Phanerochaete, Talaromyces,Chrysosporium and Penicillium are available and well known for a personskilled in the art (Sambrook et al., 1989, Penttilä et al., 1987; Jainet al., 1992; Austin et al., 1990; Bull et al., 1988; Maier et al.,2005; Akileswaran et al., 1993).

-   Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J. (1987) A    versatile transformation system for the cellulolytic filamentous    fungus Trichoderma reesei. Gene. 1987; 61:155-64.-   Jain S, Durand H, Tiraby G. (1992) Development of a transformation    system for the thermophilic fungus Talaromyces sp. CL240 based on    the use of phleomycin resistance as a dominant selectable marker.    Mol Gen Genet. 1992 September; 234(3):489-93.-   Austin B, Hall R M, Tyler B M. (1990) Optimized vectors and    selection for transformation of Neurospora crassa and Aspergillus    nidulans to bleomycin and phleomycin resistance. Gene. 1990    93:157-62.-   Bull J H, Smith D J, Turner G. (1988) Transformation of Penicillium    chrysogenum with a dominant selectable marker. Curr Genet. 1988 May;    13(5):377-82.-   Maier F J, Malz S, Lösch A P, Lacour T, Schäfer W. (2005)    Development of a highly efficient gene targeting system for Fusarium    graminearum using the disruption of a polyketide synthase gene as a    visible marker. FEMS Yeast Res. 2005 April; 5(6-7):653-62.-   Akileswaran L, Alic M, Clark E K, Hornick J L, Gold M H. (1993)    Isolation and transformation of uracil auxotrophs of the    lignin-degrading basidiomycete Phanerochaete chrysosporium. Curr    Genet. 1993; 23(4):351-6.

By “gene” is meant here in particular a gene or genes encodingregulatory proteins, such as transcription factors, othertranscriptional regulators, protein kinases, proteins involved inhistone modification or chromatin remodelling, or genes located nearcellulase or hemicellulase genes (co-expressed) in the genome of afilamentous fungus host. The genes have been selected by using themethod as described herein. The function of the genes has beenexemplified in Trichoderma, in particular in T. reesei which show theeffect of these genes in protein production. Modification of the genesin other filamentous fungi, in particular Aspergillus will be useful forimproved protein production. The “gene” in the present invention ispreferably a Trichoderma gene. Within the scope of the present inventionare also the closest homologues of the genes in other species offilamentous fungi and nucleotide sequences hybridizing under stringentconditions to said genes or said homologues. Within the scope of thepresent invention are also fragments, derivatives or other nucleotidesequences of said genes hybridizing under stringent conditions to saidgenes or said homologue. The “gene” may be isolated, which means that itis isolated from its natural components. The “gene” may be partly orcompletely synthetic. Within the scope of the present invention are alsoderivatives of said gene, which refer to nucleic acid sequencescomprising deletions, substitutions, insertions or other modificationscompared to said gene, but having the same or equivalent function assaid gene.

A “fungal host” denotes here any fungal host strains selected orgenetically modified to produce (or not produce) efficiently a desiredproduct and is useful for protein production for e.g. analytical,medical or industrial use. A fungal host is in particular “a fungalproduction host” that is suitable for industrial production of certainprotein products. The host strain is preferably a recombinant strainmodified by gene technological means to efficiently produce a product ofinterest. The fungal host may belong for example to Trichoderma,Aspergillus, Fusarium, Neurospora, Talaromyces, Phanerochaete,Chrysosporium or Penicillium genera. Typically the host is Trichodermaor Aspergillus host.

The “closest homologue of a Trichoderma gene” in other species offilamentous fungi means here a gene that has the highest percentage ofidentical nucleotides with the Trichoderma gene of all the genes of theorganism; or a gene whose protein product has the highest percentage ofidentical amino acids with the protein product encoded by theTrichoderma gene of all the gene products of the organism. The sequenceidentity of homologous regulatory genes in different organisms istypically very low. Typically, the sites binding either to DNA or otherprotein factors involved in the regulation event share homology, but theintervening sequences between these sites may not be conserved.Therefore the total % of sequence identity of homologous regulatorygenes in different organisms remains relatively low. However, thepercentage of sequence identity in the aligned nucleotide sequence canbe used as a measure to identify the closest homologue of the gene inthe other organism, thus a likely functional counterpart of the gene inthe other organism. Software and algorithms for homology searches aswell as public databases with whole genome sequence information for avariety of species exist, such as the BLAST program (Stephen F.Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, ZhengZhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402. Reference for compositional scorematrix adjustment: Stephen F. Altschul, John C. Wootton, E. MichaelGertz, Richa Agarwala, Aleksandr Morgulis, Alejandro A. Schaffer, andYi-Kuo Yu (2005) “Protein database searches using compositionallyadjusted substitution matrices”, FEBS J. 272:5101-5109) and the NCBIdatabase(http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism=fungi).

A specific “gene” is here represented by a specific sequence (SEQ IDNO)”. The effect of the gene has been shown by using the sequence of aspecific SEQ ID NO (which is here a Trichoderma sequence). The sequencemay comprise additional sequence in front of and/or after the codingregion of the gene. As described here, instead of the Trichodermasequence, the closest homologue from another filamentous fungus could beused. As is known to a person skilled in the art, also sequenceshybridizing under stringent conditions to the Trichoderma sequence or toits closest homologue, could be used.” wherein stringent conditionsrefer here to an overnight incubation at 42 degree C. in a solutioncomprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 ng/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65 degree C.

The detection methods of cellulase activity, CBHI activity or EGIactivity are well known for a person skilled in the art. The methods aredescribed for example in Bailey and Tähtiharju, 2003; Collen et al.,2005; van Tilbeurgh et al., 1982, 1985, 1988. Collett, A., Saloheimo,M., Bailey, M., Penttilä, M. & Pakula, T. M. (2005) Protein productionand induction of the unfolded protein response in Trichoderma reeseistrain Rut-C30 and its transformant expressing endoglucanase I with ahydrophobic tag. Biotech. Bioeng. 89, 335-344. Bailey M J, Tähtiharju J.2003. Efficient cellulase production by Trichoderma reesei in continuouscultivation on lactose medium with a computer-controlled feedingstrategy. Appl Microbiol Biotechnol 62:156-62.van Tilbeurgh H,Claeyssens M, de Bruyne C. 1982. The use of 4-methylumbelliferyl andother chromophoric glycosidases in the study of cellulolytic enzymes.FEBS Lett 149:152-156.van Tilbeurgh H, Loontiens F G, de Bruyne C K,Claeyssens M. 1988. Fluorogenic and chromogenic glycosides as substratesand ligands of carbohydrates. Methods Enzymol 160:45-59.van Tilbeurgh H,Pettersson G, Bhikabhai R, De Boeck H, Claeyssens M. 1985. Studies ofthe cellulolytic system of Trichoderma reesei QM 9414. Reactionspecificity and thermodynamics of interactions of small substrates andligands with the 1,4-beta-glucan cellobiohydrolase II. Eur J Biochem148:329-34.

The cultures of the strains modified for the regulatory genes can beanalysed for the produced protein pattern more in detail using a broaderset of enzyme activity measurements specific for different enzymes. Inaddition, the produced protein pattern can be analysed using 2D gelelectrophoresis followed by identification of the proteins by massspectrometry. The 2D gel analysis can reveal also quantitativedifferences in the produced protein patterns between the cultures of thedifferent strains. This information can reveal specific sets of producedproteins that are affected by the specific genetic modification.Furthermore, additional genetically modified strains can be constructedto be able to analyse the effects of both the overexpression and thedeficiency (typically deletion) of the gene, and by these means toreveal the target genes and proteins whose function is affected by themodification of the gene, and to demonstrate the effect of the specificgene modification on the produced protein pattern. These geneticallymodified strains can also be subjected to transcriptional profiling inorder to elucidate the effect of the genetic modification on the geneexpression levels and to reveal target genes affected by themodification. The information of the effects of the genetic modificationon the produced protein pattern (from the enzyme assays and 2D gelanalysis of the culture supernatants) as well as information on thetranscriptional responses caused by the genetic modification, and thetarget genes for the regulatory events it is possible to modify theprotein production properties and efficiency, as well as the compositionof the protein mixture produced into the culture medium in a definedway.

The improvement in protein production process can mean either increasedproduction of all the secreted proteins, or improved production of aspecific set of proteins, or reduced production of unwanted products, ora faster or shorter production process for the protein, or a productionprocess with less consumption of nutrients for other products thanproteins (e.g. for biomass, or unwanted side-products), or betterphysico-chemical properties protein producing unit (e.g. less viscosecultivation, better morphology of the production host), or betterdown-stream processing properties of cultivation and the proteinproduct, or better physico-chemical quality of the product.

According to specific embodiments of the invention one or more of thegene sequences tre66966 (SEQ ID NO:1), tre112524 SEQ ID NO:2), tre123668(SEQ ID NO:3) and tre120120 (SEQ ID NO:5) (Table 2); or the closesthomologue of at least one of said sequences in Trichoderma, Aspergillus,Fusarium, Neurospora, Chrysosporium, or Penicillium; or a fragment orderivative of any of said genes, or other sequence hybridizing understringent conditions to at least one of said sequences or saidhomologues, are overexpressed in a fungus host. The overexpression ofthese genes in the genetically modified host causes increased productionof cellulase, hemicellulase, other proteins involved in degradation oflignocellulosic material and/or other proteins, typically secretedproteins and/or production of total extracellular protein as compared tothe parental host, or proteins produced using promoters of genesencoding secreted proteins. This can be detected either as highermaximal level of enzymatic activity or protein produced during thecultivation or as a higher level of produced enzymatic activity orprotein produced at any time point of cultivation resulting in higherproduction level at earlier stages of cultivation and thus in fasterproduction process as compared to parent host.

Cellulase activity can be measured e.g. as enzymatic activity againstthe substrate, 4-methylumbelliferyl-β-D-lactoside (MULac). Methods formeasuring the combined activity of CBHI, EGI and β-glucosidase (referredhere as “total MULac” activity), as well as the separate activities ofthe enzymes, using MULac as substrate have been described by Bailey andTähtiharju, 2003; Collen et al., 2005; van Tilbeurgh et al., 1982, 1985,1988. The hemicellulase activity can be measured e.g. as activityagainst the birch xylan substrate (Bailey et al., 1992, Bailey M. J.,Biely, P. and Poutanen, K. (1992) Interlaboratory testing of methods forassay of xylanase activity. J. Biotechnol. 23: 257-270), and productionof total extracellular protein by using any of the methods formeasurement of protein concentration known in the art, for example usingBio-Rad Protein Assay (Bio.Rad).

It is advantageous, if one or more of genes tre66966 (SEQ ID NO:1),tre112524 SEQ ID NO:2), tre123668 (SEQ ID NO:3), still more preferablyeither one or both of genes tre66966 (SEQ ID NO:1) and/or tre112524 SEQID NO:2), are overexpressed in a fungus host. The term “gene”encompasses here also the closest homologue of at least one of saidsequences in Trichoderma, Aspergillus, Fusarium, Neurospora,Talaromyces, Phanerochaete, Chrysosporium or Penicillium; or a fragmentor derivative of said genes or other nucleotide sequence hybridizingunder stringent conditions to at least one of said sequences or saidhomologues.

TABLE 1 Plasmid T. reesei gene strain pMH35 tre66966 35-10 pMH17tre112524 17-6  pMH18 tre123668 18-11 pMH22 tre120120 22-1 

According to other specific embodiments of the invention gene tre112524(SEQ ID NO:2); or the closest homologue of said sequence in Trichoderma,Aspergillus, Fusarium, Neurospora, Chrysosporium or Penicillium; or afragment or derivative of said genes or other nucleotide sequencehybridizing under stringent conditions to said sequence or saidhomologues, is overexpressed in the fungus host. The overexpression ofthis gene causes increased cellulase activity, in particular enzymeactivity against MULac substrate under conditions measuring specificallyEGI activity. The overexpression of this gene causes also increasedhemicellulase activity. This can be detected as increased production.Furthermore, the overexpression this gene causes increased production ofextracellular protein in the culture medium.

Also other combinations of the mentioned genes can be overexpresedand/or made deficient in a fungus host to get a combined effect of thegenes on protein production properties of the fungus host. Inactivationor reduced activity of one or more of mentioned genes may be beneficialto reduce production of unwanted side-products. Within the scope ofprotection are thus genetical modification of a fungus host byoverexpression or by making deficient at least one of said genes.Overexpression of the above mentioned genes in filamentous fungus hostswas exemplified here by constructing Trichoderma strains expressingthese genes. The expression of the same or corresponding genes i.e.closest homologues of said genes, in other filamentous fungi hosts, iswithin the scope of protection of the present invention.

The corresponding genes may also be either overexpressed or madedeficient, as described above, also in other filamentous fungi hosts.

In some other applications it is desirable to tailor a protein product,in which for example higher levels of EGI and xylanase activity aredesired. For that application, the gene tre112524 (SEQ ID NO:2); or theclosest homologue of said gene in Trichoderma, Aspergillus, Fusarium,Neurospora, Chrysosporium or Penicillium; or a fragment or derivative ofsaid gene or other nucleotide sequence hybridizing under stringentconditions to said gene or to at least one of said homologues, should beoverexpressed in the fungus host.

Also sequences hybridizing under stringent conditions to the Trichodermagenes or their closest homologues in other filamentous fungus hosts arewithin the scope of protection of the present invention. In thefollowing text, by “gene” is meant also the closest homologue of thegene or sequences hybridizing into said gene or said homologue as hereindescribed. By “gene” is in the following text meant also any fragment orderivative or modified form comprising deletions, substitutions,insertions or other genetic modifications, but having the same orequivalent function as the said “gene”.

To exemplify the effect of the genes on protein production, the geneswere overexpressed in T. reesei QM9414 host as described in the Examples4-6.

The structure of the genes is similar in that they all comprise asequence domain typical to genes encoding regulatory proteins, such astranscription factors as shown in Table 2.

TABLE 2 IPR001183, Fungal transcriptional IPR007219, regulatory FungalIPR000182, protein, N- transcriptional IPR001680, GCN5-related N- GeneID terminal factor W D40 repeat acetyltraansferase tre123668 pMH 18 xtre66966 pMH 35 x tre120120 pMH 22 x tre112524 pMH 17 x x

The effect of gene tre66966 (FIG. 1), represented by SEQ ID NO:1, wasexemplified by constructing strain T. reesei 35-10 overexpressing thegene. The overexpression of this gene caused increased cellulaseproduction measured as total Mulac activity, CBHI activity and EGIactivity. In particular CBHI activity was increased compared to theparent host. Also hemicellulase production, measured as production ofxylanase activity, and total extracellular protein production wereincreased as compared to the production by the parent host strain. Theoverexpression of gene tre66966 can be used in particular to increasecellulase or hemicellulase production, or the production of othersecreted proteins. In addition, production of various heterologousproteins under the promoters of cellulase or hemicellulase genes (orsimilarly regulated promoters) can be produced at higher level by thestrains overexpressing the gene as compared to the parent host strain.

The effect of gene tre112524 (FIG. 2), represented by SEQ ID NO:2, wasexem-plified by constructing strain T. reesei 17-6 overexpressing thegene. In the cultures of the strain 17-6, the maximal level of totalextracellular protein produced was reached earlier than in the culturesof the parent host strain. By overexpression of the gene a fasterpro-duction process for secreted proteins can be obtained. Specifically,the strains 17-6 produced higher amount of hemicellulase activity,measured against xylan substrate, as well as a slightly higher amount ofcellulase activity, measured as total MULac activity or EGI activity.However, production of CBHI activity was not affected by theoverproduction Improved production of hemicellulase activity or selectedcellulase activities can be obtained by overexpressing the gene 112524.In the overexpressing strain also improved production of heterologousproteins under the promoters of these hemicellulase and cellulase genepromoters can be obtained.

The effect of gene tre123668 (FIG. 3), represented by SEQ ID NO:3, wasexemplified by constructing strain T. reesei 18-11 overexpressing thegene. The overexpression of this gene caused increased cellulaseproduction, measured especially as total Mulac activity or CBHIactivity, as compared to the parent host. The overexpression of the genehad also slightly positive effect on the production of hemicellulaseactivity, measured as xylanase activity. In the cultures of the strain18-11, the maximal level of the produced activities were higher andreached earlier than in the cultures of the parent host. Theoverexpression of the gene had also slightly positive effect on theproduction of the total extracellular protein, especially when theamount of produced protein per fungal biomass was inspected. Theoverexpression of gene tre123668 can be used in particular to increasecellulase or hemicellulase production, or the production of othersecreted proteins. In addition, production of various heterologousproteins under the promoters of cellulase or hemicellulase genes (orsimilarly regulated promoters) can be produced at higher level by thestrains overexpressing the gene as compared to the parent host strain.

The effect of gene tre120120 (FIG. 5), represented by SEQ ID NO:5, wasexemplified by constructing strain T. reesei 22-1 overexpressing thegene. The overexpression of the gene had a positive effect on theproduction of cellulase activity, measured as total MULac activity, orespecially as CBHI activity. In the cultures of the strain 22-1, themaximal level of the produced activities were reached earlier than inthe cultures of the parent host. Also the production of thehemicellulase activity, measured against xylan substrate, reached themaximum level faster than in the cultures of the parent host strain. Thecultures of 22-1 produced less biomass than the cultures of the parenthost strain. The improved faster production of proteins in the culturemedium during cultivation as compared to the parent host was especiallypronounced when the amount of produced amount of protein or enzymeactivity per fungal biomass was inspected. The overexpression of thegene had also a positive effect on the production of the totalextracellular protein when the amount of produced protein per fungalbiomass was inspected, especially at the earlier stages of thecultivation. By overexpressing gene tre120120, it is possible to improvethe production of cellulases and hemicellulases, or other secretedproteins. Also production of heterologous proteins produced under thepromoters of cellulase or hemicellulase genes can be improved byoverexpression of the gene. Especially, by overexpression of the gene itis possible to make the production process faster. Also less nutrientsis consumed for biomass formation in the strain overproducing the gene.As the improvement in production was more pronounced in the case of CBHIactivity production as compared to EGI activity production,overexpression of the gene can be also used to preferentiallyoverproduce CBHI activity or heterologous proteins under cbh1 promoter.

The effect of the genes as described herein has been exemplified byoverexpressing them in Trichoderma. The strain was T. reesei QM9414(ATCC 26921), which is generally available to the public.

Total Mulac is measured as enzyme activity against Mulac substratemeasuring activities of CBHI, EGI, and BGL.

EGI has been measured as enzyme activity against Mulac in the presenceof glucose, to inhibit BGLI, and cellobiose, to inhibit the activity ofCBHI

CBHI has been measured as enzyme activity against Mulac obtained bysubtracting the activity measured in the presence of glucose andcellobiose from the activity measured in the presence of glucose and inthe absence of cellobiose.

The methods for determining cellulase activities has been described inBailey and Tähtiharju, 2 003; Collen et al., 2005; van Tilbeurgh et al.,1982, 1985, 1988.

EXAMPLES Example 1 Cultivation of Trichoderma reesei for TranscriptomeAnalysis to Study the Cellular Responses During Induction of HydrolyticEnzyme Production Cultivation Procedure

Trichoderma reesei Rut-C30 (ATCC56765) was cultivated in shake flasks inmedium containing 7.6 g/l (NH₄)₂SO₄, 15.0 g/l KH₂PO₄, 2.4 mM MgSO₄.7H₂O,4.1 mM CaCl₂.H₂O, 3.7 mg/l CoCl₂, 5 mg/l FeSO₄.7H₂O, 1.4 mg/lZnSO₄.7H₂O, 1.6 mg/l MnSO₄.7H₂O, and 10 g/l sorbitol. pH of the mediumwas adjusted to 4.8 by addition of KOH. The cultures were inoculatedwith 8×10⁷ spores/200 ml medium and grown for 4 days in conical flasksat 28° C. with shaking at 250 rpm. For induction of the hydrolyticenzyme production, the cultures were combined and aliquots of thecombined culture transferred to flasks containing inducing medium (200ml of the culture per 90 ml of the inducing medium). The composition ofthe inducing medium was as described above except for containing 2 g/lsorbitol and supplemented either with Avicel cellulose, pretreated wheatstraw, pretreated spruce or sophorose. Uninduced control cultures weretreated similarly except that no supplement was used in the inducingmedium. The concentrations of the inducing substances were 1% (w/v) ofAvicel, wheat or spruce or 0.7 mM sophorose. Pretreatment of spruce andwheat straw was done using steam explosion, followed by washing steps.The fibrous fraction of the material was used for the induction.

Collection of Samples and Sample Treatment

Samples for analysis of biomass production, pH of the culturesupernatant, and for RNA isolation were collected at different timepoints during the pre-cultivation step as well as from the combinedculture before induction. After addition of the inducing substances thecultures were sampled for pH measurement and RNA isolation; biomassformation was measured only from separate uninduced control flasksreserved for the purpose. The sampling time points of the inducedcultures were 0 h, 6 h, 17 h, and 41 h after the onset of induction.Biomass dry weight was measured by filtering and drying mycelium samplesat 105° C. to constant weight (24 h). For RNA isolation mycelium samplesof 50 ml were filtered, washed with equal volume of 0.7% (w/v) NaCl,frozen immediately in liquid nitrogen, and stored at −80° C. Total RNAwas isolated using the Trizol™ Reagent (Gibco BRL) essentially accordingto manufacturer's instructions, and purified further by columnpurification (Qiagen, manufacturer's instructions). cDNA was synthesisedfrom the purified RNA, followed by fluorescent labelling and expressionmicroarray analysis using custom oligonucleotide microarrays by RocheNimbleGen, Inc. The design for the microarray probes and slides was doneaccording to T. reesei genome version v2.0(http://genome.jgi-psf.org/Trire2/Trire2.home.html)

Monitoring of the Cultures

During the precultivation stage the progress of the cultivations wasmonitored by analysis of biomass formation and change of pH in theculture supernatant. After the onset of the induction biomass formationwas only measured from uninduced control cultures specifically dedicatedfor the purpose, since the insoluble material in the inducing mediacould not be seoarated from fungal biomass. pH was measured from all thecultures throughout the cultivation procedure. Biomass dry weight (g/l)in the precultures before induction and in the uninduced controlcultures are sgown in FIG. 7A and pH of the cultures in FIG. 7B. Thebiomass and pH data show that, at the induction time point 100 h, thecultivations were actively growing and growth continued during theinduction time period in the control cultures. The extent of pH decreasein the cultures during time suggest equal growth characteristics of thereplicate cultures. No significant difference was detected between theuninduced and sophorose induced cultures either. In cultures withAvicel, the pH decreased slightly faster as compared to the uninducedculture, and the cultures with spruce and wheat showed fastest decreasein the pH, the difference being, however, relatively small.

Example 2 TRAC Analysis of a Selected Set of Genes Encoding HydrolyticEnzymes

In order to select the optimal time points of the induction experimentfor the expression microarray analysis, transcription levels of a set ofknown genes encoding hydrolytic enzymes were analysed using TRAC method.The relative expression levels are shown for abf1 (arabinofuranosidase1), bga1 (beta-galactosidase 1), bgl1 (beta-glucosidae 1), bxl1(beta-xylosidase 1), cip1 (cellulose-binding), cip2 ( ) egl1(endoglucanase 1), girl (glucuronidase 1), man1, xyn2 and xyn4. Clearinduction was detected for majority of the genes at the time points 6 hand 17 h, and also at 41 h of the sophorose cultures, and these timepoints were selected for the microarray analysis. The transcript levelsdetected by the TRAC analysis are shown in FIG. 8A and FIG. 8B.

Example 3 Expression Microarray Analysis of the Induced Cultures

The cultures induced either with Avicel, sophorose, pretreated wheatstraw or pretreated spruce were subjected to microarray expressionanalysis. The time points 0 h, 6 h, 17, and 41 h of the uninduced andsophorose induced cultures were used for the analysis, and the timepoints 0 h, 6 h and 17 h were selected for the Avicel, wheat and spruceinduced cultures. The microarray analysis was done using customoligonucleotide microarrays by Roche NimbleGen, Inc. The design for themicroarray probes and slides was done according to T. reesei genomeversion v2.0 (http://genome.jgi-psf.org/Trire2/Trire2.home.html). Rawmicroarray data was analysed using R and the Bioconductor packagesOligo, Limma and Mfuzz.

The analysis showed co-expression of a group of genes together withcellulase or hemicellulase genes. These genes included novel previouslynot described genes with sequence domains typical to genes for differenttypes of regulatory proteins, including genes for transcription factors,kinases, and proteins involved in histone modification and chromatinremodelling, phosphatidylinositol/phosphatidylcholine transfer protein.In order to evaluate the effect of these genes on protein production,and specifically production of hydrolytic enzymes, like cellulases andhemicellulases, a set of these genes were cloned and overexpressed in T.reesei QM9414 (ATCC 26921). The selected genes had a significantlyhigher expression at least in three of the inducing conditions studiedas compared to the uninduced cultures at the same time point, or thatthe expression profile of the genes was similar to the expressionprofiles of known hemicellulase or cellulase genes (according to M fuzzclustering of the expression data). The selected genes with theircorresponding protein identity number, predicted functional predictionbased on the sequence domains, and information on their induction in thepresence of different inducing substances at different time points ofthe induction experiment are listed in Table 3.

The information provided on the genes include the ID number according toJGI genome version 2.0(http://genome.jgi-psf.org/Trire2/Trire2.home.html), predicted functionof the gene based on the sequence data, and data on induction of thegene in the presence of different inducers (Avicel, pretreated wheatstraw, pretreated spruce or sophorose) at different induction timepoints. Statistically significant induction (higher expression level ascompared to the uninduced control cultures at the same time point) inthe Avicel, Spruce, Wheat or Sophorose induced cultures at the timepoints 0 h, 6 h, 17 h, or 41 h is indicated by “1”, and statisticallysignificant reduction in the expression level by “−1”.

TABLE 3 The induced genes encoding putative regulatory factors andselected for overexpression in T. reesei. Avicel Avicel Avicel WheatWheat Wheat Gene ID Class Extension 0 h 6 h 17 h 0 h 6 h 17 hTRIRE0066966 Regulation G-protein. 0 0 0 0 0 0 beta. WD-40 repeatTRIRE0112524 Regulation Transkcription 0 0 0 0 0 0 factor TRIRE0123668Regulation GCN5-related 0 1 0 0 1 1 N- acetyltransferase TRIRE0120120Regulation GCN5-related 0 1 1 0 1 1 N- acetyltransferase Spruce SpruceSpruce Sophorose Sophorose Sophorose Sophorose Inducer Gene ID 0 h 6 h17 h 0 h 6 h 17 h 41 h Time (h) TRIRE0066966 0 0 0 0 0 0 0 TRIRE01125240 0 0 0 0 0 0 TRIRE0123668 0 1 1 0 1 1 1 TRIRE0120120 0 0 1 0 0 1 1

Example 4

The primers used for amplification of the genes from T. reesei QM6a(CBS383.78) genome were as follows:

The 5′end primer contained a general part consisting of four 5′terminalG's, 25 nt attB1 site (ACAAGTTTGTACAAAAAAGCAGGCT) (SEQ ID NO: 6) and a 8nt region upsam from start codon of cbh1 gene (TGCGCATC), altogetherforming the sequence GGGGACAAGTTTGTACAAAAAAGCAGGCTTGCGCATC (SEQ ID NO:7)

The general component of the oligo was followed by gene specificsequence corresponding to the 5′end of the gene.

The 3′end primer contained four 5′terminal G's, 25 nt attB2 site

(SEQ ID NO: 8) (ACCACTTTGTACAAGAAAGCTGGGT)and the nucleotides CTTA followed by the gene specific sequencecorresponding to the 3′end of the gene.

The gene specific part of the primers were designed based on the ORFprediction in the genome version v2.0(http://genome.jgi-psf.org/Trire2/Trire2.home.html), or in some cases,as indicated below, according to the genome version v1.2(http://genome.igi-psf.org/trire1/trire1.home.html)

The primers used for amplification of the genes using genomic T. reeseiDNA as a template were the following:

pMH17 112524 (SEQ ID NO: 9)5′GGGGACAAGTTTGTACAAAAAAGCAGGCTTGCGCATCATGGCTGGATC GCCTGCTGCTG pMH17 112524 (SEQ ID NO: 10)3′GGGGACCACTTTGTACAAGAAAGCTGGGTACACATTCATCCCTGCGCC CAG  pMH18 123668(SEQ ID NO: 11) 5′GGGGACAAGTTTGTACAAAAAAGCAGGCTTGCGCATCATGCCTCTCGTTGTCGTCCCAG pMH18 123668 (SEQ ID NO: 12)3′GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATTGAGCAGCGGCTC GCG  pMH22 120120(SEQ ID NO: 13) 5′GGGGACAAGTTTGTACAAAAAAGCAGGCTTGCGCATCATGTCCCGCCAAATCTCCCACC pMH22 120120 (SEQ ID NO: 14)3′GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACTCGGTGCTGATACT TCT  pMH35 66966(SEQ ID NO: 17) 5′GGGGACAAGTTTGTACAAAAAAGCAGGCTTGCGCATCATGGCCAAGAAGGCGCGTC pMH35 66966 (SEQ ID NO: 18)3′GGGGACCACTTTGTACAAGAAAGCTGGGTGCTAGGCGCCGTTGACGAC  TC

PCR amplification reaction using the primers mentioned above resulted inDNA fragments containing the gene specific sequences described belowinserted between the 5′ and 3′ terminal sequences originating from thegeneral parts of the primers.

Example 5 Construction of T. reesei Strains Overexpressing theRegulatory Genes Selected Based on the Transcriptome Data from theInduction Experiment on Avicel, Wheat, Spruce or Sophorose ContainingMedia

The genes encoding putative regulatory factors co-expressed with knowncellulase or hemicellulase genes were amplified by PCR from theTrichoderma genome (T. reesei QM6a; CBS383.78) and cloned into anexpression vector which was then transformed into T. reesei QM9414. Thetransformants were selected based on function of the AmdS selectionmarker gene present in the expression cassette, transformants werepurified from colonies originating from single spores, and theintegration of the expression cassette into the genome was confirmed byPCR amplification of the cassette. Schematic view of the plasmids usedfor transformation is shown in FIG. 8.

Example 6 Cultivation of the T. reesei Strains Overexpressing thePutative Regulatory Factors, and Analysis of the Cultures for Growth andProtein Production

The modified strains overexpressing genes encoding the putativeregulatory factors were cultivated in shake flasks on medium containing7.6 g/l (NH₄)₂SO₄, 15.0 g/l KH₂PO₄, 2.4 mM MgSO₄.7H₂O, 4.1 mM CaCl₂.H₂O,3.7 mg/l CoCl₂, 5 mg/l FeSO₄.7H₂O, 1.4 mg/l ZnSO₄.7H₂O, 1.6 mg/lMnSO₄.7H₂O and supplemented with 4% lactose and 2% spent grain extract.The cultures were analysed for growth and protein production, includingassays for cellulase and hemicellulase activity. Cellulase activity wasmeasured using 4-methylumbelliferyl-β-D-lactoside (MULac) as asubstrate. The MULac can be used for measurement of combined activitiesof CBHI, EGI and BGL in T. reesei cultures (Bailey and Tahtiharju, 2003;Collen et al., 2005; van Tilbeurgh et al., 1982, 1985, 1988. Using amodified method MULac substrate can used also to measure the activitiesof CBHI and EGI specifically (Bailey and Tahtiharju, 2003; Collen etal., 2005; van Tilbeurgh et al., 1982, 1985, 1988). Xylanase activitywas measured using birch xylan as a substrate (Bailey M. J., Biely P.,and Poutanen, K., (1992) Interlaboratory testing of methods for assay ofxylanase activity. J. Biotechnol. 23: 257-270). For the results onprotein production in the cultures of the modified strains and theparental strain T. reesei QM9414, see the FIGS. 1-5.

Example 7

The closest homologue of a gene in another fungal species can beidentified e.g. based on homology searches against genome databasesusing programs such as Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi).The homology searches can be done using either the nucleotide sequenceof the gene or the amino acid sequence of the translated gene as aquery. Information on the closest homologue of the gene can be obtainedin any organism with a sequenced genome. Complete genome sequences areavailable for homology searches for a multitude of fungal species, andthe number of fully sequenced fungal organisms is still increasing.

In this example, the translated sequences of the ORFs of the T. reeseigenes tre77513, tre80291, tre41573, tre74765 and tre64608 were subjectedto BLASTP homology search against protein sequence databases of a set offungal species. The translated sequences of the T. reesei ORFs wereaccording to JGI (Joint Genome Institute; genome version 2.0,http://genome.jgi-psf.org/Trire2/Trire2.home.html).

The search was carried out using the fungal genome BLAST databases atNCBI (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism=fungi)containing completed genome sequence and whole genome shotgun sequencedata with the corresponding protein sequences of a large number offungal species. The databases used in the search in this example were:

-   -   Completed Aspergillus fumigatus proteins;    -   Completed Aspergillus nidulans FGSC A4 proteins;    -   Completed Aspergillus terreus proteins;    -   Completed Chaetomium globosum CBS 148.51 proteins;    -   Completed Gibberella zeae PH-1 proteins;

(The reference for the BLASTP 2.2.23+ program used: S. F. Altschul, T.L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J.Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Res. 25:3389-3402. Referencefor compositional score matrix adjustment: S. F. Altschul, J. C.Wootton, E. M. Gertz, R. Agarwala, A. Morgulis, A. A. Schaffer, andY.-K. Yu (2005) “Protein database searches using compositionallyadjusted substitution matrices”, FEBS J. 272:5101-5109.).

The parameters used in the searches were as follows:

General Parameters:

-   -   Max target sequences: 100 (Maximum number of aligned sequences        to display (the actual number of alignments may be greater than        this)).    -   Short queries: Automatically adjust word size and other        parameters to improve results for short queries.    -   Expect threshold: 10 (Expected number of chance matches in a        random model)    -   Word size: 3 (The length of the seed that initiates an        alignment)

Scoring Parameters:

-   -   Matrix: BLOSUM62 (Assigns a score for aligning pairs of        residues, and determines overall alignment score)    -   Gap Costs: Existence:11, Extension:1 (Cost to create and extend        a gap in an alignment)    -   Compositional adjustments: Conditional compositional score        matrix adjustment (Matrix adjustment method to compensate for        amino acid composition of sequences)

Filters and Masking:

-   -   Filter: Low complexity regions filter (Mask regions of low        compositional complexity that may cause spurious or misleading        results).

Table 4 shows examples of the closest homologues of the T. reesei genesin other fungal species obtained by the BLAST search: the closesthomologue of tre66966 in G. zeae, tre112524 in A. nidulans, tre123668 inC. globosum and tre120120 in P. marneffei. fumigatus.

TABLE 4 Examples of the closest homologies of the T. reesei genes inselected fungal species based on the BLAST search *)Identical The bestalignment amino Identical Query acids amino Alignment sequence peracids/ Identities length/ length Blast query length in the query Query(amino Blast E length of the alignment length sequence acids) Hitsequence Species Score value (%) alignment (%) (%) translated 994ref|XP_384527.1|, Gibberella 979 0.00E+00 67 492/686 71 69 tre66966FG04351.1 zeae translated 949 ref|XP_664000.1|, Aspergillus 142 5.00E−3315 111/392 28 41 tre112524 AN6396.2 nidulans translated 212ref|XP_001220518.1|, Chaetomium 88 2.00E−17 31  66/208 31 98 tre123668CHGG_01297 globosum translated 222 gb|EEA27105.1| Penicillium 48.10.00002 20  45/158 28 71 tre120120 marneffei

1. A method to genetically modify a filamentous fungus host for improvedprotein production, said method comprising genetically modifying afilamentous fungus host to overexpress with increased amount oractivity, or to be deficient with reduced or lacking amount or activityof one or more genes selected from the group consisting of genestre66966 (SEQ ID NO:1), tre112524 SEQ ID NO:2), tre123668 (SEQ ID NO:3)and tre120120 (SEQ ID NO:5); or of the closest homologue of at least oneof said genes in Trichoderma, Aspergillus, Fusarium, Neurospora,Talaromyces, Phanerochaete, Chrysosporium or Penicillium; or of afragment or derivative of any of said genes or other nucleotide sequencehybridizing under stringent conditions to at least one of said genes orsaid homologues, said host being capable of increased or decreasedproduction of cellulase, hemicellulase, other proteins involved indegradation of lignocellulosic material and/or other proteins ascompared to the parental strain.
 2. The method according to claim 1,wherein the filamentous fungus host is genetically modified tooverexpress one or more of the genes selected from the group consistingof genes tre66966 (SEQ ID NO:1), tre112524 SEQ ID NO:2), tre123668 (SEQID NO:3) and tre120120 (SEQ ID NO:5); or the closest homologue of atleast one of said genes in Trichoderma, Aspergillus, Fusarium,Neurospora, Talaromyces, Phanerochaete, Chrysosporium or Penicillium; orof a fragment or derivative of any of said genes or other nucleotidesequence hybridizing under stringent conditions to at least one of saidgenes or said homologues, said host being capable of increasedproduction of cellulase, hemicellulase, other proteins involved indegradation of lignocellulosic material and/or other proteins ascompared to the parental strain.
 3. The method according to claim 1,wherein the filamentous fungus host is genetically modified to bedeficient of one or more of the genes selected from the group comprisinggenes tre66966 (SEQ ID NO:1), tre112524 SEQ ID NO:2), tre123668 (SEQ IDNO:3) and tre120120 (SEQ ID NO:5); or of the closest homologue of atleast one of said genes in Trichoderma, Aspergillus, Fusarium,Neurospora, Talaromyces, Phanerochaete, Chrysosporium or Penicillium; orof a fragment or derivative of any of said genes or other nucleotidesequence hybridizing under stringent conditions to at least one of saidgenes or said homologues, said host being capable of decreasedproduction of cellulase, hemicellulase, other proteins involved indegradation of lignocellulosic material and/or other proteins ascompared to the parental strain.
 4. The method according to claim 1,wherein the host is selected from the group consisting of Trichoderma,Aspergillus, Fusarium, Neurospora, Talaromyces, Phanerochaete,Chrysosporium and Penicillium.
 5. A filamentous fungus host geneticallymodified to overexpress or to be deficient of one or more genes selectedfrom the group consisting of genes tre66966 (SEQ ID NO:1), tre112524 SEQID NO:2), tre123668 (SEQ ID NO:3) and tre120120 (SEQ ID NO:5); or of theclosest homologue of at least one of said genes in Trichoderma,Aspergillus, Fusarium, Neurospora, Talaromyces, Phanerochaete,Chrysosporium or Penicillium; or of a fragment or derivative of any ofsaid genes or other nucleotide sequence hybridizing under stringentconditions to at least one of said genes or said homologues, said hostbeing capable of increased or decreased production of cellulase,hemicellulase, other proteins involved in degradation of lignocellulosicmaterial and/or other proteins as compared to the parental strain. 6.The host according to claim 5, wherein the filamentous fungus host isgenetically modified to overexpress one or more genes selected from thegroup consisting of genes tre66966 (SEQ ID NO:1), tre112524 SEQ IDNO:2), tre123668 (SEQ ID NO:3) and tre120120 (SEQ ID NO:5); or of theclosest homologue of at least one of said genes in Trichoderma,Aspergillus, Fusarium, Neurospora, Talaromyces, Phanerochaete,Chrysosporium or Penicillium; or of a fragment or derivative of any ofsaid genes or other nucleotide sequence hybridizing under stringentconditions to at least one of said genes or said homologues, said hostbeing capable of increased production of cellulase, hemicellulase, otherproteins involved in degradation of lignocellulosic material and/orother proteins as compared to the parental strain.
 7. The host accordingto claim 5, wherein the filamentous fungus host is genetically modifiedto be deficient of one or more genes selected from the group consistingof genes tre66966 (SEQ ID NO:1), tre112524 SEQ ID NO:2), tre123668 (SEQID NO:3) and tre120120 (SEQ ID NO:5); or of the closest homologue of atleast one of said genes in Trichoderma, Aspergillus, Fusarium,Neurospora, Talaromyces, Phanerochaete, Chrysosporium or Penicillium; orof a fragment or derivative of any of said genes or other nucleotidesequence hybridizing under stringent conditions to at least one of saidgenes or said homologues, said host being capable of decreasedproduction of cellulase, hemicellulase, other proteins involved indegradation of lignocellulosic material and/or other proteins ascompared to the parental strain.
 8. The host according to claim 5,wherein the host is selected from the group consisting of Trichoderma,Aspergillus, Fusarium, Neurospora, Talaromyces, Phanerochaete,Chrysosporium and Penicillium.
 9. The host according to claim 5, whereinthe host is a filamentous fungus production host.
 10. A method forimproved production or for producing an improved composition of proteinsin a filamentous fungus host, said method comprising a step ofcultivating the modified filamentous fungus host of claim 5 undersuitable culture conditions for protein production.
 11. The methodaccording to claim 10, wherein the protein is selected from the groupconsisting of cellulases, hemicellulases, side chain cleaving enzymes,lignocellulose degrading enzymes, pectinases, ligninases; amylolyticenzymes; proteases; invertases; phytases, phosphatases, andhydrophobins.
 12. The method according to claim 10, wherein the proteinis a heterologous or recombinant protein and expressed under a promoterof a gene encoding any one of the proteins whose production is effectedby genetical modification of the host.
 13. The method according to claim12, wherein the the promoter is a promoter of a gene encoding acellulase, a hemicellulase, other protein involved in the degradation oflignocellulosic material or other secreted protein.
 14. The methodaccording to claim 12, wherein t the promoter is a promoter of a geneencoding a protein selected from the group consisting of cellulases,hemicellulases, side chain cleaving enzymes, lignocellulose degradingenzymes, pectinases, ligninases; amylolytic enzymes; proteases;invertases; phytases, phosphatases and hydrophobins.